Continuous flow ultracentrifuge



March 3, 1970 c. H. CHERVENKA ETAL 3,498,531

commuous mow ULTRACENTRIFUGE 2 Sheets-Sheet 1 Filed Feb. 16. 1968INVENTORS CHARLES 1-1 CHERVENKA BY KAREN s. CHERRY HG 4 I ATTORNEY 2sheets-shjeet '2 m V 5 Y I: %///////////fi//// IN CH ES H. CH VENKA BY KEN s. c RY ATTORNEY M;arch 3, 19-70 c. H. CH EVIEN-KA Filed Feb. 16,v.1968

3,498,531 CONTINUOUS FLOW ULTRACENTRIFUGE Charles H. Chervenka,Sunnyvale, and Karen S. Cherry, Los Altos, Calif., assignors to BeckmanInstruments, Inc., a corporation of California Filed Feb. 16, 1968, Ser.No. 706,043 Int. Cl. B04b 11/00, 7/04, N02

US. Cl. 233-12 Claims ABSTRACT OF THE DISCLOSURE A predetermined volumeof gas, such as air, is introduced into a rotor assembly of a continuousflow centrifuge immediately prior to the introduction of a high densitydisplacement liquid to prevent mixing between such displacing liquid andthe density gradient solution containing the centrifuged particles ofinterest as the displacing liquid forces the density gradient solutionfrom the rotor cavity. The predetermined air volume lodges in a verticalslot provided in the rotor core which slot communicates by way of aconnecting radial slot with the outer surface of the core memberdisposed in the cavity to prevent the displacing liquid from reachingthe rotor cavity through this passageway. As a result, all of thedisplacing liquid enters the rotor cavity at the outside edge of thecavity, thus preventing mixing of the displacthe liquid and the densitygradient solution containing the particles of interest.

BACKGROUND Continuous flow or zonal centrifugation has become anincreasingly important analytical tool in the investigation of thenature of numerous biological and other chemical substances. Typically,in continuous flow centrifugation a sample solution is continuouslypumped through a rotor cavity which is filled with an appropriatedensity gradient solution. During centrifugation the particles ofinterest disperse in a radial direction throughout the density gradientsolution and at equilibrium are suspended in the density gradientsolution at a location wherein their respective buoyant densitiescorrespond to that of the solution.

To remove the particles of interest the density gradient solutioncontaining the centrifuged particles is displaced from the rotor cavityby means of a liquid having a density greater than the highest densityportion of the density gradient solution. Ideally, all of the displacingliquid is fed to the outer edge of the rotor cavity. However, inpractice some of the displacing liquid flows through the sample exitpassageway provided in the rotor core and enters the rotor cavity at thesurface of the rotor core member. Since the lighter density portions ofthe density gradient solution are located closest to the surface of thecore member, mixing between the displacing liquid and the densitygradient solution takes place (as the displacing liquid gravitates undercentrifugal force toward the outer edge of the rotor cavity) causingsubstantial dilution of the rotor contents. Such mixing, of course, ishighly undesirable and, in fact, limits the usefulness of a continuousflow centrifuge for certain experimental investigations.

One approach to the solution of this mixing problem has been to insert aflexible O-ring in the vertical portion of the sample exit passagewayprovided in the core, which O-ring acts as a valve to control the flowof fluid through this passageway. The operation of the O-ring valve isgoverned by the particular rotational speed of the rotor. That is, atlow rotational speeds the elastomeric O-ring valve is closed to blockthe flow of liquids through the sample exit passageway. On the otherhand, at high ro- United States Patent 0 3,498,531 Patented Mar. 3, 1970ICC" tational speeds the O-ring valve is subjected to a suflicientcentrifugal force to cause the valve to open and allow the flow of fluidthrough the passageway. Thus, it is apparent that when a flexible O-ringvalve is employed the sample solution may be pumped only at rotorrotational speeds above the minimum required to activate the valve andthe heavy unloading solution may be pumped only when the rotor isrotated at speeds below this minimum speed. Of course, the particularspeed required to activate the O-ring valve may vary with use as theproperties of the O-ring change. Moreover, replacement of the O-ringrequires the substitution of a second O-ring of very similar propertiesand siZe which, from a design standpoint, is extremely diflicult toachieve to say the least.

SUMMARY The present invention contemplates a method for use in acontinuous flow ultracentrifuge for preventing the inadvertent mixing ofthe displacing liquid and the den sity gradient solution containing thecentrifuged particles of interest as the displacing liquid forces thedensity gradient solution out of the rotor cavity. To this end, apredetermined volume of gas, such as air, is introduced into the rotorassembly immediately prior to the introduction of the heavy densitydisplacing liquid. The predetermined volume of air lodges in a portionof the sample exit passageway provided in the top of the rotor core toblock the flow of the displacing liquid through this passageway and,thus, prevents the mixing of the displacing liquid with the densitygradient solution contained in the rotor cavity. In an alternativeembodiment contemplated by the present invention an additional radialslot is provided in the core member to receive the predetermined volumof air which blocks the flow of the displacing liquid through the sampleexit passageway.

Accordingly, a primary object of the present invention is a new andnovel method for preventing the inadvertent mixing between thedisplacing liquid and the density gradient solution contained in therotor cavity of a continuous flow centrifuge.

Another object is the provision of a method for preventing theinadvertent mixture between the displacing liquid and the densitygradient solution contained in the rotor cavity, which method isindependent to the rotational speed of the rotor.

Another object is a simple and expedient method of preventing theintermixing of a displacing liquid and a density gradient solutioncontained in the rotor cavity of a continuous flow centrifuge.

These and other objects and advantages of the present invention willbecome apparent from the following detailed description when read inconjunction with the ac 'co'mpanying drawings in which:

FIG. 1 is an elevation view, partly in section, showing a continuousflow centrifuge apparatus;

FIG. 2 is an enlarged partial sectional view showing a suitablecentrifuge rotor;

FIG. 3 is a reduced schematic perspective view of the rotor core;

FIG. 4 is a plan view of the rotor core taken along lines 4-4 of FIG. 3.

With reference now to the drawings and more particularly to FIG. 1thereof, it will be observed that the continuous flow ultracentrifugeincludes an outer housing 10 which serves to enclose a drive means,designated generally by the reference numeral 2, and a rotor housing 3.The top of outer housing 10 is provided with an opening 5 through whicha rotor 6 may be passed for mounting within a rotor chamber 7 on a driveshaft or spindle 8. A movable door 9 threadably receives and supports anupper bearing and seal assembly 11. Door 9 is provided with spacedrollers 12 which ride in space channels 13 secured to the sides ofhousing to provide easy access to chamber 7. A latch mechanism (notshown) cooperates in conjunction with controls (not shown) to releasablylock door 9.

The side walls of rotor chamber 7 include a generally cylindrical steelmember 14 which acts as a guard should the rotor core explode under thestrain created by the relatively high rotational velocities at which itis operated. The lower end of member 14 includes a bottom wall 15likewise made of relative strong material. The interior of rotor chamber7 may be provided with refrigeration means including evaporator coils 16which serve to control the temperature therein.

A suitable vacuum seal is formed between bottom wall 15 and spindle 8 asit extends upwardly into chamber 7. Preferably spindle 8 is made offlexible material and extends downwardly with its lower end journaled inan oil filled bearing assembly 17. Bearing assembly 17 is supported byspace brackets 18 extending downwardly from a resiliently mounted base19. Base 19 may be supported from a fixed member 2 by springs 21. Adriven pulley 22 is carried by spindle 8 and is driven by a belt 23 froma drive pulley 24 and motor 25. Means (not shown) may be disposed inhousing 10 for evacuating air from chamber 7.

The rotor assembly 40, as best illustrated in FIG. 2, includes acylindrical bowl 26, the wall of which is threaded at both ends 27 and28. End 28 threadably receives a closure member or base 29 whichincludes a generally cylindrical well 31 adapted to receive the upperend of drive spindle 8. Drive spindle 8 fits tightly into well 30 toprovide a positive frictional drive connection between spindle 8 andbase member 29 whereby rotor bowl 26 may be driven.

The rotor assembly, designated generally by the reference numeral 40,also includes a rotor core member 33 which fits tightly over a boss 34provided at the upper end of base 29. Boss 34 is provided with an O-ring31 to provide a tight frictional interconnection between core member 33and base 29 so that the core member 33 rotates with base 29.

The upper end of bowl 26 threadably receives a cover or lid member 35. Asuitable O-ring 36 is interposed between a shoulder 37 formed on thesides of bowl 26 and the lower edge of lid member 35 while a secondO-ring 32 is held in an annular groove formed in the top of core member33 to provide an effective seal between core 33 and lid member 35. Thelid member 35 is positioned with respect to the rotor core 33 to providea relatively large annular space 38 between the upper end of the rotorcore 33 and the lid member 35. The lid member 35 is also provided wtih aplurality of radially extending, downwardly sloping bores or holes 39which communicate between the annular space 38 and the outer edge 41 ofthe rotor chamber. In practice, four equally spaced holes 39 are formedin lid member 35. However, it will be appreciated that, due to thenature of the illustrated cross-sectional view, only one such hole (39)is shown.

A feed and shaft assembly 42 extends downwardly through an apertureprovided in cover member 35 into a well 43 provided in the top of rotorcore member 33. The shaft assembly 42 includes an outer tubular conduit44 and a concentric inner tubular coduit 45 each of which is adapted tocarry fluids into and out of the rotor chamber simultaneously duringrotation.

The rotor core 33 is a solid cylinder, formed from a suitable metal,which is tapered slightly in an outward direction from bottom to top andwhich includes at least four projecting vanes, 46, 47, 48 and 49 (bestshown in FIG. 3) that divide the rotor cavity into four sectorcompartments for minimizing turbulence. Core member 33 is provided witha small centrally located axial hole 50 extending from top to the bottomof the core member 33 and which communicates with the rotor cavity 100by way of a radial extending, downwardly sloping, hole 51 formed in thebottom of the rotor core 33. In practice, rotor core 33 is provided withfour equally spaced holes 51 but, again, due to the nature of theillustrated crosssectional view, only one hole 51 is shown. The innerfluid-carrying tube 45 communicates with the axial hole formed in thecenter of the rotor core while the outer fluid-carrying tube 44cooperates with both the annular space 38 provided at the top of thecore 33 and the radial holes 39 formed in the cover member 35 which leadto the outer edge 41 of rotor cavity 100.

Core member 33 is also provided with four equally spaced verticalextending holes 53 in the top of the core which cooperate with fourradially extending slots 54 extending to the surface 55 of core member33 disposed in rotor cavity 100. Each radial slot 54 communicates with avertical hole 53 to form a sample eflluent exit passageway.

The inner fluid-carrying tube 45 cooperates with an outlet tube 56 whilethe outer fluid-carrying tube 44 is associated with a second outlet tube57.

To facilitate a complete understanding of the operation of the presentinvention it is believed it would first be appropriate to discussbriefly the typical operating procedure used in analyzing suitablesamples by continuous flow ultracentrifugation techniques. Initially therotor is loaded by pumping a density gradient solution into the rotorcavity by way of outer tubular line 44, annular space 38 and downwardlysloping radial hole 39 formed in the cover member 35. The lightestdensity portion of the density gradient solution is pumped into therotor cavity first and enters at the outer edge 41 of the rotor cavity100. As successively denser portions of the gradient are pumped into therotor cavity 100 they in turn force the lighter density portions of thedensity gradient solutions centripetally toward the surface 55 of thecore member 33. After the gradient is in the rotor cavity, a fluidhaving a density greater than the highest density portion of the densitygradient solution, which fluid is generally referred to as a cushion, isintroduced into the rotor cavity through outer tubular feed-line 44,annular space 38 and radial holes 39 to completely fill the rotor cavity100.

After the rotor cavity has been completely filled with the densitygradient solution, the fluid flow through the outer tubular conduit 44is terminated and the sample solution is introduced into the rotorcavity 100 by way of inner tubular conduit 45, centrally located axialhole 50 and downwardly sloping radial holes 51 provided in the bottom ofcore member 33. The sample solution flows through the rotor cavity 100from the bottom to the top along the surface of core member 33, andexits from cavity 100 by Way of sample exit passageway comprising radialslot 54, vertical hole 53, and annular space 38. The sample solutionthen flows out through outer tubular conduit 44 and connecting conduit56 to a suitable container (not shown).

After centrifugation the density gradient solution containing thecentrifuged particles of interest is displaced from the rotor cavity 100by means of a highly dense liquid. That is to say, a highly dense liquidis fed to the outer edge 41 of the rotor cavity 100 by way of outertubular conduit 44, annular space 38, and radial holes 39. Thedisplacing liquid being of greater density pushes or forces the lighterdensity gradient liquid solution containing the particles of interestout of the bottom of the rotor cavity 100 through radial holes 51,centrally located axial extending hole 50, inner tubular conduit 45, andconnecting conduit 56 to a suitable fraction collector (not shown). Inpractice it has been found that some of the displacing liquid flowsthrough the sample exit passageway comprising vertical hole 53 andradial slot 54 provided in the top of the core member 33 and enters therotor cavity 100 at core surface 55. As previously discussed, inasmuchas the lighter density portions of the density gradient solution arelocated toward this surface of the rotor core member, the displacingliquid (being of a much greater density) flows in a radial directionoutwardly through the density gradient solution and, thus, intermixeswith it. This naturally results in a dilution of the density gradientsolution containing the particles of interest and detrimentally affectsthe end resolution of the ultracentrifugation process.

In accordance with the principles of the present invention, to preventthe intermixing of the displacing liquid and the density gradientsolution containing the centrifuged particles of interest, apredetermined volume of gas, such as air, is introduced into the rotorassembly immediately prior to the introduction of the displacing liquid.In particular, the predetermined volume of air is fed by way of outertubular conduit 44 to the top of the rotor assembly 40. The volume ofair which is introduced through this passageway completely fills eachvertical hole 53 provided in the top of core member 33 and partiallyfills the annular space 38 between cover member 35 and core 33. Itshould be noted that the vertical holes 53 are orientated perpendicularto the direction of centrifugal force produced by the rotor assembly andhence the volume of air tends to lodge in these vertical holes 53. Thehighly dense displacing liquid, which follows the predetermined volumeof air down the outer tubular conduit 44, easily passes through theannular space 38 to the radial holes 39 and enters the rotor cavity 100at the cavitys outer edge 41. Of course, since the displacing liquid isof much higher density than that contained in the rotor, its movement tothe outer radius is aided by the centrifugal force field. However, thevolume of air lodged in the vertical holes 53 blocks the flow ofdisplacing liquid through the sample exit passageways. It should benoted that relatively high pressures would be required to displace theair out of holes 53, because the only escape passage for this air is byway of radial slots 54, against the centrifugal field. Consequently,none of the displacing liquid may enter the rotor cavity 100 at coresurface 55 and henceintermixing between the displacing liquid and thedensity gradient solution is prevented. Moreover, the flow of the samplesolution in the normal direction (from the bottom to the top of thecore) is not hindered in the least since the sample flow rate issufficient tc sweep the air block out of the sample exit passageway.

The volume of air necessary to effectively block the sample exitpassageway while at the same time keeping free the radial holes 39leading to the outer edge 41 of rotor cavity 100 is generally governedby the particular type of rotor assembly used in the continuous flow centrifuge system. For instance, in a suitable rotor manufactured by thepresent assignee and designated model B-IX, a volume of around 2.5 to4.0 milliliters was found to be effective. Of course, it will beappreciated that the necessary volume of air varies according to thecircumstances. The volume of air may be conveniently controlled byremoving the tubular conduit 57 at the seal housing 11 and flushing itwith displacing liquid and then draining the required quantity back outof the line before re-attaching it. The length of empty tubing may thenbe used to indicate the volume of air contained therein.

In an alternative embodiment commensurate with the principles of thepresent invention, there is provided in at least one core sector anadditional slot or small bore 60 (as indicated by the dotted line) inthe top of the core member 33 immediately above the O-ring 32, whichbore extends in a radial direction to interconnect vertical hole 53 andannular space 38. That is, bore 60 communicates with a small space (notshown) between cover member 35 and the right side of the top of coremember 33 to form a generally U-shaped passageway. After forming thispassageway, each of the vertical portions 53 of the sample exitpassageways is plugged with a suitable stopper (not shown). As a result,the air being fed into the as sembly flows through this U-shapedpassageway.

In operation the predetermined volume of air introduced into the rotorassembly immediately prior to the displacing liquid lodges in each ofthe additionally formed bores 60 and thus forces the displacing liquidto flow through radial holes 39 to the outer edge 41 of rotor cavity ina manner previously discussed. It will be appreciated that either one orall four of the sample exit passageways may be modified in thisjust-described manner if desired.

Numerous modifications and departures from the specific apparatusdescribed herein may be made by those skilled in the art withoutdeparting from the inventive concept of the invention. Accordingly, theinvention is to be construed as limited only by the spirit and scope ofthe appended claims.

What is claimed is:

1. A method for use with a continuous flow centrifuge to prevent mixingbetween the displacing liquid and the density gradient solutioncontaining the centrifuged particles of interest as the displacingliquid forces the density gradient solution from the rotor cavitycomprising the steps of introducing a predetermined volume of gas intothe rotor assembly and then introducing a displacing liquid into therotor cavity containing the density gradient solution.

2. A method for use with a continuous flow centrifuge as defined inclaim 1 wherein the predetermined volume of gas is transmitted to theentrance to the rotor cavity, said predetermined volume of gas enteringthe lodging in a portion of a sample exit passageway provided in the topof a rotatable core member disposed in the rotor cavity and displacingliquid is blocked from flowing through the sample exit passageway by thepredetermined volume of gas lodged therein and thus forced to flowthrough an alternate slot communicating with the outer edge of the rotorcavity.

3. In a continuous flow centrifuge apparatus for preventing mixingbetween displacing liquid and the density gradient solution containingthe particles of interest as the displacing liquid forces the densitygradient solution from the rotor cavity comprising a generallycylindrical bowl, a cover member secured to the open end of said bowl,the interior surface of said bowl member and said cover member defininga rotor cavity, a rotor core member disposed in said cavity, means fordriving said bowl and core members together at a predeterminedrotational speed, said core member including a small bore communicatingwith the bottom portion of said rotor cavity, the top surface of-saidcore member and the interior surface of said cover member defining anannular space, the upper end of said core member including a sample exitpassageway connecting said annular space with the edge of said coremember, the cover member including a radial extending hole connectingsaid annular space with the outer edge of said rotor cavity, conduitmeans passing through an aperture in the cover member, one end of saidconduit means communicating with said annular space and means fortransmitting a predetermined volume of gas through said conduit meansimmediately prior to transmitting a displacing liquid through saidconduit means, said predetermined volume of gas becoming lodged in saidsample exit passageway to block the flow of displacing liquid throughthe sample passageway and forcing substantially all of the displacingliquid to flow through the radial hole provided in the cover member tothe outer edge of said rotor cavity to thereby prevent the intermixingof the displacing liquid and the density gradient solution containingthe centrifuged samples of interest.

4. Apparatus as claimed in claim 3 wherein said sample exit passagewayincludes a vertical orientated portion and a radially extending portion,said predetermined volume of gas being directed to and becoming lodgedin said vertical orientated portion.

5. Apparatus as claimed in claim 3 wherein said sample exit passagewayincludes a vertically orientated portion and a radially extendingportion and said core member includes a second passageway including aradially extending portion connecting said vertical portion of saidsample exit passageway to said annular space, and means for blockingsaid vertical portion of said sample exit passageway whereby saidpredetermined volume of air is directed to and becomes lodged in theradially extending portion of said second passageway.

References Cited UNITED STATES PATENTS 1,634,246 6/1927 Jones et al.233- 8 1,866,638 7/1932 Fawcett 23347 3,332,614 7/1967 Webster et a1.233-43 OTHER REFERENCES 370,651 4/1932 Great Britain.

WILLIAM I. PRICE, Primary Examiner US. Cl. X.R. 23316, 28, 46

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No.3,498,531 March 3, 1970 Charles H. Chervenka et a1.

It is certified that error appears in the above identified patent andthat said Letters Patent are hereby corrected as shown below:

Column 6, line 28, "the" should read and line 31, after "and" insert theSigned and sealed this 24th day of November 1970.

(SEAL) Attest:

WILLIAM E. SCHUYLER, JR.

Commissioner of Patents Edward M. Fletcher, J r.

Attesting Officer

